Hematology control and system for multi-parameter hematology measurements

ABSTRACT

Hematology control compositions and systems used to measure a plurality of parameters in a blood sample are provided. The hematology control compositions are particularly useful as a control for multi-parameter, automated instrument systems. The control compositions comprise a reticulocyte component, a white blood cell component, a red blood cell component, a nucleated red blood cell component, a platelet component and a reticulated platelet component. Methods of making and using the control compositions are also provided.

CLAIM OF BENEFIT OF EARLIER FILING DATE

This application is a continuation of copending application Ser. No.09/740,509 filed on Dec. 19, 2000, which is a continuation ofapplication Ser. No. 09/504,816, filed on Feb. 16, 2000, now U.S. Pat.No. 6,221,668, which is a continuation-in-part of application Ser. No.09/378,608, filed on Aug. 20, 1999, now U.S. Pat. No. 6,200,500 .

FIELD OF THE INVENTION

The present invention relates generally to hematology controlcompositions and systems and, more particularly, to a hematology controlcomposition and system used to measure a plurality of parameters in asample of blood with a multi-parameter automated hematology instrument.

BACKGROUND OF THE INVENTION

Hematology controls for various automated instruments that measure, forexample, red and white blood cell counts and platelet counts, are knownin the art and are described in the following U.S. Pat. Nos. 3,558,522;3,873,467; 4,179,398; 4,219,440; 4,299,726; 4,324,687; 4,358,394; and4,436,821 (incorporated by reference herein). Currently, blood analysisrequires the use of one or more of several different instruments and,subsequently, different blood samples and blood sample preparations toanalyze the various components of blood. Several hematology instruments,however, now have the capability of measuring various parameters ofblood without requiring separate sample preparation for each parameterbeing tested. Such instruments include the Beckman Coulter STKS or Gen-SSystems, the Abbott Cell-Dyn 4000 Hematology System, Bayer ADVIA 120,and the Sysmex XE2100 System. These improved automated instruments canmeasure one or more of: 1) reticulocytes, 2) red blood cells, 3)nucleated red blood cells, 4) platelets, 5) reticulated platelets, 6)white blood cells, including lymphocytes, monocytes, neutrophils,eosinophils, basophils, and 7) white blood cells with all phenotypes. Itwould thus be desirable to provide a hematology control composition thatcould be used as a control in connection with these instruments.

In the preferred embodiment, a hematology control composition for usewith a multi-parameter automated hematology instrument includes a liquidsuspension of particulates that has measureable characteristics likewhole blood. The control composition includes one or more blood cells(i.e. cells handled or treated to simulate such a component as found inwhole blood) or their analogs (collectively referred to as blood cellcomponents), that may or may not be fixed, stabilized, or prepared byother treatment prior to final suspension. In different embodiments, theblood cell components may be derived from a source that will exhibit thesize, shape or other measurable characteristics of human, animal, orother whole blood. By way of examples, U.S. Pat. Nos. 4,198,206;4,436,821; 5,008,021; 5,262,327; 5,270,208; 5,432,089; 5,460,797;5,672,474; 5,677,145; 5,731,205; 5,811,099 and 5,981,282, which are eachhereby incorporated by reference, each contain examples of these typesof blood cell components. The control has one or more blood component toresemble corresponding components in whole blood when measured by themulti-parameter automated hematology instrument. When so measured, thecontrol composition would assist in the calibration, operation, andaccumulation of quality assurance data for the multi-parameter automatedhematology instrument.

Also of potential interest may be U.S. Pat. No. 5,888,790 and “ImprovedIsolation of Normal Human Reticulocytes via Exploitation ofChloride-Dependent Potassium Transport,” Sorette et al., Blood, Vol. 80,No. 1 (July 1), 1992: pp. 249-254; hereby incorporated by reference.

SUMMARY OF THE INVENTION

A hematology control and system for multi-parameter hematologymeasurement is provided. The hematology control provides values for thevarious constituents of blood that the multi-parameter hematologyinstrument is capable of measuring. The hematology control compositioncomprises components for simulating reticulocyte, white blood cell, redblood cell, nucleated red blood cell, platelet or reticulated plateletconstituents of whole blood.

Methods of making and using the hematology control composition of thepresent invention are also provided herein.

The system of the present invention also includes a hematologyinstrument, a control, and may further include output or readoutdevices. In one embodiment the system includes other peripheral devices,such as a device for tracking samples and associating them withparticular data, such as a bar-code scanner system.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The hematology control composition of the present invention comprisescomponents for simulating one or more of the following constituents ofwhole blood: reticulocytes, white blood cells, red blood cells,nucleated red blood cells, platelets, or reticulated platelets. In oneembodiment the components are suspended in an isotonic medium,preferably including lipoprotein. The hematology control composition ofthe present invention provides values for various constituents of bloodthat a hematology instrument, such as a multi-parameter hematologyinstrument, is capable of measuring. Examples of multi-parameterhematology instruments include those available commercially withoutlimitation, under the designations Beckman Coulter STKS or Gen-SSystems, the Abbott Cell-Dyn 4000 Hematology System, Bayer ADVIA 120System, the Sysmex XE2100 System, or the like.

The present discussion includes multiple approaches to making a controlin accordance with the present invention, it being recognized that onehighly preferred embodiment contemplates a control having componentsthat simulate the characteristics of whole blood for purposes ofobtaining readouts on a Beckman Coulter GEN-S instrument for red bloodcells, the five populations of white blood cells, reticulocytes andplatelets. Thus, a suspension is provided that includes a plurality ofparticles that exhibit similar light scattering, conductivity,impedance, optical (including fluorescence), photometric, or otherproperty responses detected by the instrument in operation, as would thecorresponding components of whole blood.

It will be appreciated that the term “control composition” as usedherein means one or more blood components (i.e., blood constituents aswell as analogs thereof), which when combined or used alone,sufficiently simulate the relevant characteristics of whole blood forwhich the instrument tests. The following addresses the preparation ofvarious of the constituent components. The control of the presentinvention contemplates an admixture of two or more blood components, andpreferably a reticulocyte component and a component simulating at leastthree, and preferably five subpopulations of white blood cells.Percentages are by volume unless otherwise indicated.

Reticulocyte Component

The reticulocyte component of the control composition includes acomponent that exhibits the relevant characteristics for detection ofreticulocytes using a hematology instrument in accordance with thepresent invention. Accordingly, the control may suitably containstabilized reticulocytes (that is, immature anucleate red blood cellscontaining some ribonucleic acid) or an analog thereof. For example,among possible embodiments, the reticulocyte component may comprise truemammalian reticulocytes prepared for instance by mammalian (e.g. human)red blood cell encapsulation or by isolation from whole blood. Thereticulocyte component is prepared in any suitable manner. See. U.S.Pat. No. 5,432,089, incorporated by reference. Alternatively, it ispossible to obtain suitable reticulocytes by obtaining blood from ananemic animal (e.g., a pig, goat, rabbit or the like).

In one particularly preferred embodiment, the reticulocyte component isprepared by an encapsulation method, using non-reticulocyte blood cells,such as red blood cells from a human or other source. The red bloodcells are encapsulated and stabilized. By way of illustration, in oneembodiment, red blood cells having a relatively high MCV (e.g., about 85to about 95 fL, and more preferably about 88 to about 92 fL) areprovided.

The cells are washed in a suitable diluent (e.g., about 0.15 M NaCl) inone or more washing steps. The cells are then concentrated to a desiredhematocrit value, e.g., greater than about 50%, and more preferablygreater than about 70%.

The cells are then encapsulated with RNA. By way of example, RNA isencapsulated into the red blood cells by a suitable lysis step, e.g., byhypotonic lysis. This may be done in a number of ways, including bymixing red blood cells with a solution of RNA having an appropriate pHand osmolarity. For instance the solution may contain a minor amount ofRNA (more preferably about 1%), and has a pH between about 7 and 8 (morepreferably about 7.6). The solution is adjusted (e.g. with NaCl) toobtain an osmolarity of about 40 mOsm.

The red blood cells are mixed with the RNA solution in a suitableproportion, which may vary as desired. In a present preferredembodiment, the volume ratio of packed red blood cells to RNA solutionis about 0.8 to about 1.2:about 1 to about 2, and more preferably isabout 1:1.4. Before lysing, or at an early stage of the lysing, the redblood cells and the RNA solution are pre-incubated, such as by heatingto above room temperature (e.g., about 37 degrees C). The cells arelysed in the admixture, and thereafter, the red blood cells arere-sealed. By way of example, the red blood cells are re-sealed byintroducing them to a salt solution and then heating above roomtemperature (e.g., adding about 0.15 volume of about 12% NaCl solution,and then heating or annealing the cells to about 37 degrees C for aboutone hour).

The resulting mixture is poured into a separatory funnel and allowed toincubate for a suitable time, e.g., for at least about 18 hours at roomtemperature. Thereafter, cells from the bottom portion of the funnel arecollected (e.g., from about the bottom 70% or lower). The cells arewashed with a suitable diluent, such as the diluent from Table 1 or 4.Optionally, about 0.5 to about 5.0% and more preferably about 0.75% ofStreck Laboratories product number 233301, also known as STA-CELL(available from Streck Laboratories (Omaha, Nebr.)) is added. In thepresent illustrated embodiment, the cells are thus treated in a mannerthat would make them susceptible to a detection stain (e.g., methyleneblue).

In another embodiment, the reticulocyte component is fixed (such as withan aldehyde or other suitable fixative) in a lactose or other suitablediluent. A like diluent absent the fixative may also be employed forwashing.

White Blood Cell Component

In the present embodiment, the component of the control that is tosimulate the characteristics of white blood cells in whole blood isprepared from a biological material. More specifically, the material isa cellular biological material, and preferably the material includeshuman white blood cells (although blood cells from any suitable animalmay be employed). In a preferred embodiment of the present invention,the white blood cell component includes materials for replicating therelevant measurable characteristics for some or all of each of the fivewhite blood cell types, namely, lymphocytes, monocytes, neutrophils,eosinophils and basophils, and they in turn are provided at theirart-disclosed levels (see e. . Table 6).

The white blood cell component of the hematology control compositioncomprises a blood cell (e.g. white blood cell) or analog thereof,selected from the group consisting of white blood cells for variouscellular types, white blood cells for all phenotypes, and mixturesthereof U.S. Pat. Nos. 5,270,208 and 5,262,327, incorporated herein byreference, provide examples of a suitable white blood cell component(see also U.S. Pat. No. 5,529,933, hereby incorporated by reference). Ofcourse, the skilled artisan will appreciate that the subject inventionis not limited to white blood cell components prepared from only whiteblood cells. Analogs prepared from any of a variety of other souces arepossible, including but not limited to red blood cells from birds,reptiles, mammals, etc.

The cells are provided in packs (e.g., 2 to 3 packs per bottle). Thecells are treated by a series of steps for selectively lysing anyundesired blood cells present in the material as provided; for washingthe cells; and for fixing or otherwise stabilizing the cells.

Selective lysing may be accomplished in any suitable manner, forinstance, by contacting the undesired blood cells with a lysing agent.Any suitable lysing agent may be employed. Buffered halides, such asammonium chloride and Trizma Based (e.g., about 7.5 g ammonium chlorideand 2 g Tris per liter), illustrates one suitable class of lysingagents, where the undesired cells include red blood cells. Lysing isaccomplished through a series of consecutive washing steps with thelysing agent. Optionally, before the lysing, the cells are subjected toa preliminary fixing step, such as by contacting them with a suitablefixing agent, heating them or both. For instance, the cells arecontacted with a buffered antimicrobial saline solution (optionallyincluding a diluent as described in Table 4) including a suitable amountof a fixative (e.g., about 0. 11% formaldehyde).

The lysing may be done in a single step or a plurality of steps (e.g.,for about one hour, then for about 25 minutes), where after each step,the lysing agent is removed and fresh agent is introduced. After lysing,the cells are washed to remove the lysing agent. Any suitable washcomposition and technique may be employed. For instance, the aforenotedbuffered antimicrobial saline solution (absent a fixing agent) may beemployed. Using this solution the cells are washed in at least one stepand preferably two steps, wherein the cells are centrifuged at asuitable rate (e.g. about 900 rpm for about 10 minutes) (e.g., about200× g).

Prior to fixing, it may be preferable to further pre-treat the cells inan albumin-containing diluent, such as about 2% BSA in a diluent such asthat of Table 1. Preferably the diluent has a pH of about 8 and anosmolarity of about 175 to about 300 (e.g. about 215). Any suitableperiod of pre-treatment may be employed, e.g., about one hour, whenmaintained at about 6 degrees C.

The cells are fixed in any suitable manner sufficient to denature theprotein on the cell surface. The cells may be heated, contacted with afixing agent or both. The skilled artisan will recognize that, thoughthe preferred fixing agent is an aldehyde, any suitable agent(preferably in a hypotonic solution) may be used, including forinstance, those containing an aldehyde, an alcohol, a heterocyclic urea(e.g., diazolidinyl urea (known as DU), imidazolidinyl urea (known asIDU) or a mixture thereof) or a mixture thereof Among the suitablefixing agents, one particularly effective alcohol-containing agent is50% by volume—[1-methyl-2-(5-methyl-3-oxazolidinyl)-ethoxy] methoxy]methanol (e.g., NUOSEPT 145, from HULS America, Inc.), or a mixturetherof. In one present preferred embodiment, the aldehyde is selectedfrom the group consisting of formaldehyde, glutaraldehyde, and mixturesthereof.

By way of illustration, a fixing admixture is prepared to include about10 parts cells, about 20 parts distilled water, an agent for enhancingosmolarity across the cell membrane, for aiding in the formation ofclusters in scattergram population readouts or both (e.g., about 5 g/lof a sugar, such as sorbitol), an agent for helping to stabilize thereadout of monocytes (e.g., about 4% DU), and a fixing agent, such asabout 4 parts formaldehyde, and about 0.1 parts glutaraldehyde. Fixingis performed for a suitable amount of time and at a suitabletemperature. Using this fixing agent, for example, fixing is performedfor about 2 to about 3 days at about 22 degrees C (i.e., warmed prior tofixing after refrigeration).

Fixed cells are washed with a suitable rinse material (e.g., a cellstabilizer in a diluent) to remove the fixing agent. By way ofillustration, two washes are made during centrifugation at about 900 rpmfor about 10 minutes (e.g., about 200× g), in a solution including ametal halide (e.g., about 5% NaF) cell stabilizer in a diluent (e.g. adiluent having the composition as outlined in Table 2).

In another illustrative embodiment, when preparing the white blood cellcomponent for the control composition of the present invention, thecells are obtained by standard separation from whole blood or fromportion of previously fractionated whole blood containing the desiredcell population. The cells are resuspended, for instance, in a phosphatebuffered solution containing polyethylene glycol 20,000 (PEG),ethylenediamine tetraacetic acid (EDTA) and magnesium gluconate with 2%bovine serum albumin. The osmolarity of this solution is preferablysufficient to swell the white blood cells prior to fixation (e.g. about215 mosm). The cells may then be stored in this solution, e.g. at about6° C. for 1 hour.

The cells are fixed in a suitable media in order preferably to denaturethe surface or otherwise accomplish preserving the cell morphology. Toillustrate, in one embodiment, in a solution of distilled watercontaining 5 g/l sorbitol, 7.4% formaldehyde and 0.125% glutaraldehyde.Of course, other suitable fixing agents may be used in suitable amounts.In a highly preferred embodiment, the white blood cells and the fixsolution are maintained at a temperature sufficient to provide a properwhite blood cell position (e.g. between about 4° C. and 12° C.). Thefixative is added to the cells at a suitable ratio. For example, in oneembodiment, a ratio of between 10 ml of cell suspension to 24 ml of fixsolution is used. The distilled water in the fix solution swells thewhite blood cells further, while the fixative stabilizes the cellmembrane. The cells are thus left in the fixative for 2 days at roomtemperature.

After fixation, the cells preferably are washed. In one aspect, they arewashed in a phosphate buffered solution. One such solution containspolyethylene glycol, (PEG), ethylenediaminetetraactic acid (EDTA),magnesium gluconate and bovine serum albumin. Lipoprotein concentrate isadded at 150 mg/dl HDL to store the cells prior to use in order toimprove the stability of the scattergram position while the white bloodcells are waiting to be added to the other components of the controlcomposition.

It will be appreciated that white blood cells prepared as described inU.S. Pat. No. 5,459,073 (incorporated herein by reference) for flowcytometry may be employed for phenotyping. By mixing the two types ofwhite cells, both requirements can be met, i.e., white blood cells forvarious cellular types and phenotypes, as the cells prepared forphenotyping generally should not interfere with the position of otherwhite blood cells on the histograms/scattergrams.

In controls for certain instruments, white blood cells may need to bediluted or concentrated, for example, for the Cell-Dyn instruments adilution to a count of 10,000 is preferred.

Red Blood Cell Component

In the present embodiment, the component of the control that is tosimulate the characteristics of red blood cells in whole blood isprepared from a bio-compatible material. More specifically, the materialis a cellular biological material, and preferably the material includeshuman red blood cells (although analog blood cells from any suitableanimal may be employed).

The cells as provided are separated from the liquid medium orsupernatant in which it is supplied through any suitable separationtechnique, including but not limited to centrifugation, filtration, orthe like. The cells are washed in a series of one or more (e.g., 3)consecutive washing steps, pursuant to which excess supernatant isremoved. The cells preferably are washed in a (e.g. such as the diluentof Table 2) and optionally one or more additional components selectedfrom the group consisting of a cell stabilizer, an albumin, an agent forreducing the likelihood of cell hemolysis in the presence of oxygen, andmixtures thereof. In a more preferred aspect, the additional componentsare selected from the group consisting of a metal halide cellstabilizer, bovine serum albumen (BSA), an antioxidant and mixturesthereof In still a more preferred embodiment, the diluent is one such asthat of Table 4, and it will include about 0.003 to about 0.010 (andmore preferably about 0.005%) NaF, about 2% BSA, about 0.005 to about0.020 (and more preferably about 0.010%) sulfasalizine. The diluent ofTable 1 likewise may be used as desired.

Optionally, depending upon the end use and commercial considerations,one or more agents for reducing the rate of degradation is employed in asuitable amount; for example, about 0.25 to about 2.0%, and morepreferably about 0.75% of a material available from Streck Laboratories(Omaha, Nebr.) under the product number 233301 or the designation STACELL (which material may also be suitably added for one or more of theother components). Further, optionally, the cells preferably areprepared in an environment substantially free of glucose. In yet anotheroptional embodiment, the cells are fixed (e.g., by a suitable proteindenaturation step, such as by glutaraldehyde fixing) after washing andthen further washed.

The cells may be washed for any suitable period of time, and resuspended(in the same wash or a different one, e.g., one having a higherconcentration) any suitable number of times.

The skilled artisan will appreciate that the red blood cells may bewashed free of all other cellular material, such as by using a magnesiumgluconate diluent.

By way of further illustration, in another embodiment, concentrated redblood cells are provided, separated from associated supernatant, andconcentrated human red blood cell packs are suspended in a solution(e.g. phosphate buffered solution containing PEG (MW 20,000)) andallowed to settle overnight. The supernatant is then removed and 0.5%NaCl with PEG is added in an equal volume to the packed red blood cellsand allowed to set at room temperature for 4-5 hours. The supernatant isagain removed and the cells are resuspended in the NaCl solution andstored at 6° C. overnight. The packs are further checked for excessivehemolysis and removed from the inventory. The remaining packs are pooledinto batches based on the MCV's, wherein twelve to fourteen packs arecombined to make a batch. The batches are again resuspended in the NaCIsolution for about 4-5 hours at room temperature. Of course, other timesand temperatures may be employed.

Each batch is resuspended into a phosphate buffered solution containingPEG, EDTA and magnesium gluconate. The cells are allowed to settle, thesupernatant is removed and the cells are resuspended in the abovesolution with lower PEG concentrations for storage up to 90 days at 6°C.

One diluent efficient in stabilizing the red blood cells on the CoulterSTKS includes a phosphate buffered solution containing PEG, Na₂EDTA,magnesium gluconate and an antioxidant (e.g., sulfasalazine orα-tocopherol), wherein the antioxidant is added to prevent hemolysiswhen the lipoprotein is added to the control composition of the presentinvention. The final diluent also contains about 2% bovine serum albuminto improve the position of the white blood cells. After the cells havebeen washed into this diluent, STA-CELL from Streck Laboratories (Omaha,Nebr.) is added at 0.75% to the total volume of red blood cells to giveadded stability to the MCV'S.

Optionally, in certain applications, it may be desirable to fix the redblood cells, after removing excess red blood cells, such as by a slowcentrifugation. By way of example, for such an embodiment, the diluentof Table 1 is employed. Cells are washed and resuspended in the diluentto a suitable concentration (e.g. about 4×10⁶/mm³). Preferably the pH isabout 7 and there is no glucose in the suspension. Approximately one toone proportions of the cells are admixed with the diluent and a suitableamount of a fixative (e.g., about 0.007% to about 0.01% glutaraldehyde(by count)) for a suitable period and at a suitable temperature (e.g.,22 degrees C for about one or two days). The resulting cells are thenwashed a plurality of times (e.g., about 3 to about 8) in a like diluent(preferably at a pH of between 7 and 8). The cells are centrifuged atabout 1500 for about 15 minutes. Decanting and sonication is performedas needed. Moreover, the cells may be further treated as desired by theaddition of a suitable amount of STA-CELL (e.g., about 0.75%) fromStreck Laboratories, Inc. (Omaha, Nebraska).

Nucleated Red Blood Cell Component

When employed, the nucleated red blood cell component of the controlcomposition of the present invention comprises nucleated red blood cellsor an analog thereof, such as avian red blood cells, e.g., turkey orchicken red blood cells. For example, turkey red blood cells are washedinto a phosphate buffered solution and set to a count of about1×10⁶/mm3. The cells are fixed with a phosphate solution (volume equalto the cell volume) +0.4% V/v glutaraldehyde, at room temperature forone day and then washed into a phosphate buffer.

The fixed turkey red blood cells are added to the control composition ofthe present invention to yield a cell count equal to at least 10% of thewhite blood cell count in order to produce NRBC flags on the CoulterSTKS or the Cell-Dyne 4000 manufactured by Abbott Laboratories. Thoughthe present example contemplates the use of turkey cells, cells fromother cell sources may be employed as the skilled artisan willappreciate.

Platelet Component

In a preferred embodiment of the present invention, the hematologycontrol composition additionally provides a platelet component,preferably a simulated platelet component. Among other possible types,the platelet component may comprise stabilized human platelets orplatelets simulated from goat, bovine or porcine blood cells. In oneembodiment, they are prepared from red blood cells. See, U.S. Pat. Nos.4,160,644 and 4,198,206, incorporated herein by reference, disclose anexample of a suitable platelet reference control and methods ofpreparation. The skilled artisan will appreciate a number of othertechniques for preparing simulated platelets.

In general, how the platelets are prepared may depend upon the source ofthe cells (i.e., whether they are animal blood cells to be shrunken,swollen or otherwise sized or shaped to resemble platelets, or whetherthey are platelets from blood). In general, the cells are washed,optionally pre-fixed, sized and shaped, and then fixed or otherwisestabilized in terms of size and shape, and neutralized.

By way of example, the platelet component is prepared from animal bloodcells, such as goat red blood cells. The cells are washed one or moretimes (e.g., about three times) in a suitable buffered solution such asa buffered saline solution (e.g., phosphate buffered saline solution).The solution may also include a suitable amount of a chelating agent(e.g., about 1% ethylene diamine tetraacetic acid (EDTA)).

The cells optionally are prefixed in a suitable manner to aid in thestep of sizing and shaping them. By way of illustration, the cells arecontacted with a suitable pre-fix solution ((e.g., one to one in theinitial wash solution having a fixative (e.g., about 0.0085% ofglutaraldehyde)). The amount of such fixing agent of course may besuitably adjusted as needed to control the sizing rate (e.g., theshrinkage rate). Preferably the pre-fix solution is warmed to anelevated temperature (e.g., about 30 degrees C), and prefixing isperformed for a sufficient time at such temperature (e.g., about 90minutes). After the pre-fix step, the supernatant is removed, such as bycentrifuging, aspirating or both.

In instances where the sizing and shaping is performed to shrink thecells and form a simulated platelet structure, the cells preferably arelysed in a suitable manner (e.g., using a lytic agent such as ammoniumchloride tris). During lysing, the cell size and shape may be monitoredusing a suitable instrument such as the H3 by Bayer Corporation (ahematology analyzer with a laser optical detection system). The cellsare then washed one or more times with a suitable diluent (e.g., thediluent of Table 3).

Like with the aforenoted blood cells, the simulated platelets are fixedin any suitable manner, preferably one that will denature the protein onthe cell surface. In a particularly preferred embodiment, the cells arefixed in a one to one solution with a diluent such as that of Table 3and about 0.1% formaldehyde. Preferably the temperature of the fixingsolution is elevated (e.g. about 37 degrees C) for a sutiable period oftime (e.g., about 3 days).

The cells are then washed with a suitable wash to remove the fixative,preferably also taking measures to neutralize any unreacted fixativethat has not bound to the cell surface. To illustrate, preferably wherethe fixative is an aldehyde, a suitable amount of a glycine solution isemployed in the wash. One or more additional washing steps may beperformed, using one or more additional washes, as desired. Forinstance, subsequent washing steps may be employed using the washcomposition of Table 3, Table 4 or a mixture thereof.

It will be appreciated that the platelet component can be made fromhuman blood, using any suitable process. By way of example, withoutlimitation, human blood is provided (preferably in a diluent having afixative (e.g., a diluent such as that of Table 4 with about 0.10%formaldehyde). Optionally, red blood cells are removed, and theresulting cells are admixed with fixed cells. For instance, the redcells are centrifuged in quantities of about 400 ml per container forabout 10 minutes at about 900 rpm).

As-provided cells are placed in a fixative (e.g., about one to oneproportions in a diluent such as that of Table 3 with a suitable amountof a fixative (e.g., about 0.075% glutaraldehyde)). Fixing is done atabout 22 degrees C. for about 2 days. Following fixation, centrifugationis performed at about 1800 rpm for about 20 minutes (for 400 mlcontainer). The two separate collections of cells are brought togetherand washed (such as in a 1× diluent like that of Table 1), and thencentrifuged (e.g. at about 1800 rpm for about 20 minutes). Optionally,the platelets are decanted from residual red blood cells. Sonication maybe used as desired to address platelet clumping. Decantation may also beused as desired to assure debris and red blood cells are removed.

Resulting materials are then resuspended in a diluent. An example of asuitable diluent would be that of Table 1 having no glucose and a pH ofabout 7.

Reticulated Platelet Component

In another embodiment of the present invention, the control compositioncomprises a reticulated platelet component. To illustrate, withoutlimitation, goat red blood cells with encapsulated nucleic acids wouldconstitute one example of a reticulated platelet component useful forthe present invention. Other analogs may be used as well.

By way of further illustration, in another embodiment, reticulatedplatelets for the control composition of the present invention areprepared by inducing a porous blood cell membrane to permit entry of RNAinto a cell, hemoglobin to leave, or both. The then cells are sized orshaped and stablizied. To illustrate, goat red blood cells are placed ina solution of about 0.9% NaCl and concentrated to 70-80% hematocrit(HCT). Equal volumes of concentrated red blood cells and 4% RNA solution(20 mls of each) adjusted to 300 mosm with a suitable salt (e.g., KCl)are mixed together and dialyzed against 500 mls of a hypotonic solution,such as one containing glycerol (osm=90-100), for 90 minutes at 6° C.Dialysis is required to slowly change the osmolarity without damagingthe cells. The resulting osmolarity change in the red blood cellsolution is from about 300 mosm to about 150 mosm. This process createsholes in the cell membrane to allow the RNA in the red blood cellsolution to enter the red blood cells.

The osmolarity is brought back to isotonicity by dialyzing the red bloodcells containing RNA against an isotonic solution. This dialysis is atroom temperature for 30 minutes and the final osmolarity of the cells isabout 260 mosm. This process reseals the holes that were created by thehypotonic dialysis, thus trapping the RNA inside the cells.

Eighty milliliters of the resealing diluent containing 0.1% Nuosept 101is added to the encapsulated red blood cells and the mixture is heatedat 37° C. for 3 hours. This heating step helps to lyse the weakenedcells from the encapsulation process and anneals the membranes of theencapsulated red blood cells.

To illustrate, goat red blood cells are separated from otherconstituents of goat whole blood. For instance, cells are washed intoPBS three times to remove the plasma and white cells. The concentrationis adjusted to 8×10⁶/mm3 and fixed with a volume of PBS equal to thecells that contain 0.224-0.320% glutaraldehyde, providing the amount ofprotection needed to allow proper Iysis during the shrinkage step. Thecells are incubated at 30° C. for one hour and centrifuged at 1200 RPMfor 15 minutes. The supernatant is removed and the cell volume isadjusted to one-fourth of fixed volume.

An ammonium chloride solution is added to the cells to equal theoriginal volume of fixed cells. Without intending to be bound by theory,the ammonium chloride solution creates holes in the membrane to allowhemoglobin to exit to the cells, while the glutaraldehyde protects fromtotal lysis. The cells are monitored for hemoglobin loss based on (MPV)decreases on a Bayer H-1. When the MPV is at 10 fl on the Bayer H-1, thecells are diluted with a phosphate buffered solution and centrifuged at1800 RPM for 20 minutes. The supernatant is removed and the cells arewashed to remove the free hemoglobin and shrink the membrane around thehemoglobin to produce an MPV of approximately 10 fl on impedanceinstruments, such as the S+IV, manufactured by Beckman Coulter.

When the MPV is at 10 fl on the H-1, the cells are diluted with aphosphate buffered solution and centrifuged at 1800 RPM for 20 minutes.The supernatant is removed and the cells are washed to remove the freehemoglobin and shrink the membrane around the hemoglobin to produce anMPV of approximately 10 fl on impedance instruments, such as the S+IV,manufactured by Beckman Coulter.

In a more preferred embodiment, to prevent further loss of hemoglobinand shrinkage of the membrane, the cells are fixed more than once, forinstance, with 0.04% glutaraldehyde in a volume of phosphate bufferedsolution equal to the cell volume at a count of 1×10⁶/mm3. The cells areleft at room temperature overnight and then washed. Though the presentexample contemplates the use of goat cells, cells from other cellsources may be employed (such as stabilized human platelets) as theskilled artisan will appreciate.

Suspension Medium

The present invention relates to a method of making a hematology controlcomposition for use with multi-parameter systems, comprising the step ofmixing one or more of a reticulocyte (retic) component, a white bloodcell component, a red blood cell component, a nucleated red blood cellcomponent, a platelet component, and a reticulated platelet component inan isotonic suspension medium.

The components of the control preferably are suspended in appropriateconcentrations a suitable suspension medium that permits the control tobe processed through the automated instrument. The suspension mediumthus has a pH of about 6.5 to about 8.5 and is isotonic. By way ofexample, among the possible embodiments of the present invention, theisotonic suspension medium may comprise a buffer, antioxidant, proteinor a mixture thereof, e.g., magnesium gluconate/ethylene diaminetetraacetic acid (EDTA)/phospate buffer with nucleated red blood cells;the same buffer with the additives HDL, sulfasalazine and alphatocopherol; or the same buffer with 3% albumin.

Lipoprotein

While BSA in any diluent present improves the white blood cell positionon the scattergram, lipoprotein is also preferably used in an amounteffective to provide a scattergram that represents whole blood,including the proper positioning of the five subpopulations of whiteblood cells. See U.S. Pat. Nos. 5,270,208 and 5,262,327 incorporated byreference. A lipoprotein source, preferably one consisting essentiallyof high-density lipoprotein (i.e., HDL) is added at about 0.5 to about8.0% by volume of the control, and more preferably at about 100-175mg/dl to the control composition and a-Tocopherol is further added tothe lipoprotein source to reduce peroxides produced by the oxidation ofthe lipoproteins. An example of a suitable commercially available formof lipoprotein is SUPERTRATE (available from Bayer).

Admixing Components

Stock volumes of the constituent components are prepared in thefollowing approximate concentrations:

RBC: 6.0 × 10⁶/mm³ WBC: 150,000/mm³ Platelets: 10 × 10⁶/mm³ Retics: 50%of 5.5 × 10/mm³ red count NRBC: 0.5 × 10⁶/mm³

To prepare the final control composition, for example, in a 5 litervolume, stock volumes of the constituent components are combined asfollows:

Approximate Approximate Target Count Volume Stock RBC: 4.5 × 10⁶/mm³3,750 ml WBC: 8.0 × 10³/mm³ 266 ml Plt (platelets) 225 × 10³/mm³ 112 mlRetic (%)   3% 370 ml NRBC 0.01% 4.5 ml

The combined constituents are brought to a final total volume of 5liters by adding a suitable final diluent (e.g., prepared according toU.S. Pat. No. 5,262,327, incorporated herein by reference (preferablyincluding SUPERTRATE or a like substance)). The skilled artisan willappreciate that there are other means and procedures to prepare this andother embodiments of the present invention. Moreover, concentrations canbe varied to provide controls having predetermined abnormal readingswhen tested.

Using Control

The following discusses examples of methods of using the controlcomposition to determine the accuracy and reproducibility of theoperation of a multi-parameter automated hematology instrument. By wayof example, a multi-parameter automated hematology instrument, such as aBeckman Coulter STKS or Gen-S Systems, the Abbott Cell-Dyn 4000Hematology System, Bayer ADVIA 120, and the Sysmex XE2100 System, isprovided, optionally with a slide preparation module. The claimedcontrol composition is obtained or prepared which includes, by way ofexample, a treated stabilized human red blood cell component and areticulocyte component with quality control values in an appropriaterange, for example, 1.0%, 2.5%, and 9.0%, respectively. It isrefrigerated prior to use. At the beginning of testing, the controlcomposition is allowed to warm to room temperature for about fifteenminutes, mixed manually, and checked for resuspension of contents.

The control composition is prepared and analyzed by the same standardmethod as test samples which may be tested in batch quantities by theuse of a suitable cassette having apertures for receiving test vials.After preparation, the control composition and test samples are analyzedby counting the population number of each subject component type with amulti-parameter automated hematology instrument, which will yield avisual display of the data.

For a Coulter System, the automated test instrument may employtechnology known generally as VCS Technology (as marketed by BeckmanCoulter). VCS generally analyzes cell samples in view of simultaneousvolume conductivity and Scatter measurements. Ordinarily, a startingsample is employed in combination with suitable reagents (which maycomprise a component of a kit) and physical agitation for lysing andcell measuring by way of flow cytometry.

Accordingly, the sample may be tested by the Coulter Principle of (DC)Impedance to measure the cell volume in an isotonic suspension.

Conductivity may be employed, for instance, by applying alternatingcurrent in the radio frequency range. Energy can penetrate the cell byshort circuiting the cell membrane's bipolar lipid layer.

Information about the cells is also possible with light scattertechniques, such as from the scatter characteristics detected from cellsin response to a coherent light source, e.g. a laser beam.

Of course, by no means is the mode of sample testing limited to theabove. As mentioned other principles may be used.

The respective population counts obtained from the analysis are comparedeither to known reference value for each component type in the controlcomposition, or by comparison of the population counts for eachcomponent types in the test sample with the corresponding values ofcomponents in the control composition. Data relating to the measurementof components in control composition and test samples is collected,monitored, stored, compared and analyzed by electronic means, such as acomputer programmed with appropriate software and containing appropriatedata file structure.

TABLE 1 Most Preferred Reagents Concentration Distilled Water 0.9 literMethyl Paraben 0.40 g/l PEG 20,000 3.00 g/l EDTA, Disodium Salt 7.04 g/lMagnesium Gluconate 3.92 g/l Sodium Phosphate Dibasic Anhydrous 2.68 g/lGlucose 6.0 g/l Sodium Hydroxide pellets 0.8 g/l Adenosine 0.25 g/lInosine 0.25 g/l Neomycin Sulfate 0.40 g/l Chloramphenicol 0.15 g/l*q.s. to 1 Liter

TABLE 2 Most Preferred Reagents Concentration Distilled Water 0.9 literMethyl Paraben 0.4 g/l PEG 20,000 3.00 g/l EDTA, Disodium Salt 11.73 g/lMagnesium Gluconate 6.53 g/l Sodium Phosphate Dibasic Anhydrous 4.47 g/lGlucose 10 g/l Sodium Hydroxide pellets 1.40 g/l Adenosine 0.25 g/lInosine 0.25 g/l Neomycin Sulfate 0.40 g/l Sodium Fluoride 0.05 g/lChloramphenicol 0.15 g/l *q.s. to 1 Liter

TABLE 3 Most Preferred Reagents Concentration Distilled Water 0.9 literMethyl Paraben 0.4 g/l PEG 20,000 3.00 g/l EDTA, Disodium Salt 16.75 g/lMagnesium Gluconate 9.33 g/l Sodium Phosphate Dibasic Anhydrous 6.39 g/lSodium Hydroxide Pellets 2.04 g/l Adenosine 0.25 g/l Inosine 0.25 g/lNeomycin Sulfate 0.40 g/l Chloramphenicol 0.15 g/l *q.s. to 1 Liter

TABLE 4 Most Preferred Reagents Concentration Distilled Water 1 literMethyl Paraben 0.40 g/l PEG 20,000 3.00 g/l EDTA, Disodium Salt 11.73g/l Magnesium Gluconate 6.53 g/l Sodium Phosphate Dibasic Anhydrous 4.47g/l Glucose 10 g/l Sodium Hydroxide pellets 1.40 g/l Adenosine 0.25 g/lInosine 0.25 g/l Neomycin Sulfate 0.40 g/l Sodium Fluoride 0.05 g/lBovine Serum Albumin 20 g/l Sulfasalazine 0.10 g/l Chloramphenicol 0.15g/l *Cholestrol Supertrate containing α-Tocopherol is added to theproduct at the time of combining the various cell types. The cholestrolsupertrate is added (2-3%) to make the α-Tocopherol concentration about5 mg % in the product.

The skilled artisan will appreciate that a number of the ingredientshave been disclosed by way of specific example, but that any of a numberof alternative ingredients at the suggested or different concentration,may be suitably substituted for such ingredients. The following Table 5illustrates examples of various alternatives, and also includes a briefdiscussion of the commonly employed concentration ranges when employedin the diluents of Tables 1-4. Though the ingredients are described byreference to a particular function, it should be appreciated that suchdiscussion is presented without intending to be bound by theory. In someinstances, the ingredient will perform a different or an additionalfunction. Moreover, the skilled artisan will appreciate that referencein Table 5 to the functions, in the context of the present preferredembodiments, could be made to select additional alternatives. Thus,there is no intention to be bound to the breadth of any specificillustrative ingredient or concentration, where it is apparent thatothers may be advantageously be employed in addition to or as asubstitute for such ingredient.

Likewise, as desired, ingredients may be deleted from the diluent. Thus,a combination of some of the ingredients may be suitably employed toachieve desired results.

Preferably, the diluent includes one or more agents that function as asurfactant, hemolysis inhibitor, red blood cell settler, MCV stabilizer,buffer, metabolite, osmolarity adjuster, antimicrobial, antifingal,protein source, positioner of white blood cell subpopulation,antioxidant, debris reducer or a mixture thereof.

TABLE 5 Preferred Range of Component Concentration concentration OthersPolyethylene Glycol 3.00 g/l 1.00-10.00 g/l PEG 8000, F68 (PEG MW20,000) Disodium EDTA 11.75 g/l 7.00-17.00 g/l Tetrasodium EDTAMagnesium Gluconate 6.50 g/l 3.5-9.5 g/l Lactose Sodium Phosphate 4.50g/l 2.5-6.5 g/l Citrate, Borate, Trizma Base Sodium Bicarbonate Glucose10.00 g/l 0-10 g/l Other sugars Adenosine 0.25 g/l 0.1-1.0 g/l InosineNeomycin sulfate 0.40 g/l 0.1-0.8 g/l strepto-,kana-mycinChloramphenicol 0.15 g/l 0.1-0.4 g/l Piperacillin Methyl Paraben 0.40g/l 0.20-1.20 g/l Other antifungals Bovine Serum Albumin 30.0 g/l0.0-60.0 g/l Other protein sources Cholesterol Supertrate 30 ml/l 20-50ml/l Triglyceride Supertrate, Cholesterol HDL Supertrate Cholesterol(all available through Bayer) Sodium Fluoride 0.05 g/l 0.0-0.50 g/lOther halides Sulfasalazine 0.10 g/l 0.0-0.50 g/l Other antioxidantsα-Tocopherol 0.05 g/l 0.0-0.30 g/l Ascorbic acid, BHT, (Vitamin E)Deferoxinilne Mesylate, Probucol, Rutin

TABLE 6 Cell Range (approximate % of white blood cells) Lymphocytes 20to 45% Monocytes  2 to 10% Neutrophils 40 to 75% Eosinophil 1 to 6%Basophils Up to 1%

Further Alternative Exemplary Embodiment

For the red blood cell component, a diluent containing Mg Gluconate andEDTA (e.g., about 3.92 g/l Mg Gluconate; 7.04 g/l EDTA-Disodium; 2.68g/l NA₂HPO₄; glucose 6 g/l; and antimicrobials (pH of 7.1 and osmolarityusing KCl of about 300)) stabilizes red cells so that red cellparameters are stable for 200 days.

The white blood cell component is prepared from fresh human white bloodcells. The cells are washed free of red blood cells and platelets usingthe above diluent. The cells are suspended in phosphate buffered salinewith an osmolarity of 280 and pH of 7.2. A solution of 20% Nuosept 145(Huls America) is mixed with the white blood cells 1:1 to give a finalconcentration of about 10% (e.g., 9.11%). The mixture is placed at atemperature of about 37° C. to about 50° C. for six days or more.Thereafter, the cells are washed one or more times (e.g., 3 times) in asuitable diluent, e.g., a diluent such as Table 1, centrifuged (e.g.,about 900 rpm for 10 minutes) and resuspended in a diluent such as inTable 1 (having no glucose and a pH of about 7).

The platelet component is prepared by washing human platelets free fromwhole blood components by low speed centrifugation (900 RPM for 10min.). The platelets are then stabilized by addition of a low level ofglutaraldehyde. The platelets are stabilized by mixing 1:1 with themagnesium gluconate diluent containing 0.075% glutaraldehyde (finalconcentration of 0.037% glutaraldehyde). After a 22° C. incubation, thecells are again washed in the diluent and are ready for use. The sameprocedure can be used for bovine or porcine.

The reticulocytes are prepared by encapsulation of yeast-RNA asdescribed in U.S. Pat. No. 5,432,089, incorporated by reference.

The white blood cell component may be set up with red blood cells forachieving differential stability, such as by maintaining them at about22° C. for about 20 days.

The final control contains the following cellular concentrations (Table7), exhibits a histogram/scattergram profile that includes a properlypositioned five-part differential of S white blood cells andreticulocytes, substantially approximating whole blood and is stable for200 days or more. This control provides such results for the Cell Dynseries, the Bayer H-3, and the Sysmex SF instruments.

TABLE 7 Values WBC % N % L % M % E % B RBC RBCo HCT MCV Low 3.65 43.141.7 11.0 1.94 2.32 3.82 3.80 30.8 80.6 Normal 7.92 55.9 31.0 10.4 1.311.50 5.01 4.97 42.8 85.5 High 19.2 59.9 23.3 14.8 1.86 .213 5.51 5.3948.6 88.2 Values MCHC RDW % R IRF PLTo PLTi MPV Low 34.9 13.1 5.38 .21267.6 69.4 10.2 Normal 35.0 13.0 2.79 .184 179.0 196.0 9.90 High 33.816.4 .736 .190 381.0 443.0 10.2

Accordingly, the foregoing discussion discloses and describes merelyexemplary embodiments of the present invention. One skilled in the artwill readily recognize from such discussion and from the accompanyingdrawings and claims, that various changes, modifications and variationscan be made therein without departing from the spirit and scope of theinvention as defined in the following claims. All patents and otherpublications cited herein are expressly incorporated by reference.

We claim:
 1. A hematology control composition comprising: a) astabilized reticulocyte component; b) a nucleated red blood cellcomponent; c) a platelet component; d) a lipoprotein; and e) a whiteblood cell component that is prepared from white blood cell analogs andis capable of exhibiting a five-part differential that correspondssubstantially with that of human whole blood, upon analysis by amultiparameter hematology analysis instrument.
 2. The controlcomposition of claim 1, wherein said analogs are provided from humanwhite blood cells.
 3. The control composition of claim 1, wherein saidanalogs are provided from red blood cells.
 4. The control composition ofclaim 1, wherein said white blood cell component has been contacted withglutaraldehyde.
 5. The control composition of claim 1, wherein saidwhite blood cell component has been contacted with formaldehyde.
 6. Thecontrol composition of claim 1, wherein said white blood cell componenthas been contacted with a heterocyclic urea.
 7. The control compositionof claim 1, wherein said white blood cell component has been contactedwith an alcohol.
 8. The control composition of claim 1, furthercomprising a red blood cell component.
 9. The control composition ofclaim 8, wherein said red blood cell component comprises human red bloodcells.
 10. The control composition of claim 8, further comprisingsimulated platelets.
 11. The control composition of claim 10, furthercomprising a reticulated platelet component.
 12. The control compositionof claim 11, wherein said reticulated platelet component comprises goatred blood cells.
 13. The control composition of claim 1, wherein saidstabilized reticulocyte component comprises reticulocytes prepared byisolation from whole blood.
 14. The control composition of claim 1,wherein said platelet component is derived from porcine cells.
 15. Thecontrol composition of claim 1, wherein said platelet component isderived from bovine cells.
 16. The control composition of claim 1,wherein said nucleated red blood cell component is derived from avianred blood cells.
 17. The control composition of claim 16, wherein saidnucleated red blood cell component is derived from turkey red bloodcells.
 18. The control composition of claim 16, wherein said nucleatedred blood cell component is derived from chicken red blood cells.
 19. Ahematology control composition comprising: a) a stabilized reticulocytecomponent; b) a nucleated red blood cell component; c) a stabilized redblood cell component; d) a platelet component; and e) a fixed andstabilized white blood cell component, suspended in a solution with alipoprotein, and being capable of exhibiting a five-part different thatcorresponds substantially with that of human whole blood, upon analysisby a multiparameter automated hematology analysis instrument.
 20. Thecontrol composition of claim 19, wherein said reticulocyte component isfrom blood of an anemic animal.
 21. The control composition of claim 20,wherein said reticulocyte component is from blood of an anemic pig. 22.The control composition of claim 20, wherein said reticulocyte componentis from blood of an anemic goat.
 23. The control composition of claim20, wherein said reticulocyte component is from blood of an anemicrabbit.
 24. The control composition of claim 20, wherein said whiteblood cell component is prepared from red blood cells.
 25. The controlcomposition of claim 20, further comprising a reticulated plateletcomponent.
 26. The control composition of claim 19 wherein saidstabilized reticulocyte component comprises reticulocytes prepared byisolation from whole blood.
 27. The control composition of claim 19wherein said platelet component is derived from bovine cells.
 28. Thecontrol composition of claim 19 wherein said nucleated red blood cellcomponent is derived from avian red blood cells.
 29. The controlcomposition of claim 28 wherein said nucleated red blood cell componentis derived from turkey red blood cells.
 30. The control composition ofclaim 28 wherein said nucleated red blood cell component is derived fromchicken red blood cells.
 31. A hematology control compositioncomprising: a) a stabilized reticulocyte component prepared fromencapsulation of red blood cells with RNA; b) a nucleated red blood cellcomponent; c) a red blood cell component; d) a platelet component; e)lipoprotein; and f) a white blood cell component that is prepared fromhuman white blood cells and is capable of exhibiting a five-partdifferential, upon analysis by a multiparameter automated hematologyanalysis instrument.
 32. The control composition of claim 31 whereinsaid stabilized reticulocyte component comprises reticulocytes preparedby isolation from goat red blood cells.
 33. The control composition ofclaim 31 wherein said platelet component is derived from porcine cells.34. The control composition of claim 31 wherein said platelet componentis derived from bovine cells.
 35. The control composition of claim 31wherein said nucleated red blood cell component is derived from avianred blood cells.
 36. The control composition of claim 35 wherein saidnucleated red blood cell component is derived from turkey red bloodcells.
 37. The control composition of claim 35 wherein said nucleatedred blood cell component is derived from chicken red blood cells. 38.The control composition of claim 31 wherein said white blood cellcomponent includes white blood cells prepared for having surfaceantigens retained.
 39. A hematology control composition comprising: a) astabilized reticulocyte component; b) a nucleated red blood cellcomponent; c) a platelet component; d) a lipoprotein; e) a stabilizedred blood cell component; and f) a white blood cell component that isprepared from human white blood cells and is capable of exhibiting afive-part differential that corresponds substantially with that of humanwhole blood, upon analysis by a multiparameter hematology analysisinstrument.
 40. The control composition of claim 39 wherein said whiteblood cell component further comprises analogs for each of: a)lymphocytes; b) monocytes; c) neutrophils; d) basophils; and e)eosinophils.
 41. The control composition of claim 40 wherein saidanalogs are present in amounts corresponding to: a) lymphocytes in anamount of about 20to 45 % of said white blood cell component; b)monocytes in an amount of about 2to 10 % of said white blood cellcomponent; c) neutrophils in an amount of about 40to 75 % of said whiteblood cell component; d) basophils in an amount up to about 1to saidwhite blood cell component; and e) eosinophils in an amount of about 1to6 % of said white blood cell component.
 42. The control composition ofclaim 40 wherein said white blood cells include white blood cellsprepared for having surface antigens preserved.
 43. The controlcomposition of claim 42 wherein said white blood cells include whiteblood cells prepared for having surface antigens preserved.
 44. Thecontrol composition of claim 40 wherein said nucleated red blood cellcomponent is derived from avian red blood cells.
 45. The controlcomposition of claim 40 wherein said reticulocyte component is derivedfrom true mammalian reticulocytes.
 46. The control composition of claim45 wherein said reticulocyte component is prepared by encapsulation ofred blood cells with RNA.
 47. The control composition of claim 46wherein said platelet component is reticulated platelets.
 48. Thecontrol composition of claim 46 wherein said platelet component isderived from goat red blood cells.
 49. The control composition of claim40 wherein said reticulocyte component is an analog derived from theblood of an anemic animal.
 50. The control composition of claim 39wherein said white blood cell component includes aldehyde-fixed humanwhite blood cells.
 51. The control composition of claim 50 furthercomprising a preservative agent.
 52. The control composition of claim 39wherein said white blood cell component is fixed with an aldehyde,alcohol, heterocyclic urea or a mixture thereof.
 53. The controlcomposition of claim 39 wherein said white blood cell component is fixedwith an alcohol-containing agent that is 50 % by volume or a mixturethereof.